JPWO2006025411A1 - Liposomes for improved intracellular drug delivery - Google Patents

Abstract

[PROBLEMS] To provide a drug delivery system that retains in the circulating blood and enhances the drug efficacy in the target organ and cells of the drug. SOLUTION: Polyethylene glycol cholesteryl ether is used as a part of the liposome-forming base to form the liposome. Therapeutic drug-containing liposomes using liposomes prepared using a base. Further, by adopting polyethylene glycol diacylglycerol ester as a part of the liposome-forming base, it is possible to obtain a good medicinal effect.

Description

The present invention relates to an agent for improving target intracellular delivery of a drug comprising a liposome prepared using polyethylene glycol cholesteryl ether as part of a liposome-forming base and the liposome. The present invention further relates to an agent for improving target intracellular delivery of a drug comprising a liposome prepared by adding polyethylene glycol distearyl ester as part of a liposome-forming base and the liposome.

Stealth liposomes that stay in the circulation have been reported for the purpose of sustaining bioactive substances or therapeutic compounds in the body or increasing the distribution of drugs, especially anticancer drugs and antibacterial drugs, to target organs (
, , ). Stealth liposomes mean liposomes that avoid interactions with blood components, blood cells, and vascular endothelial cells by coordinating a water-soluble polymer such as polyethylene glycol (PEG) on the liposome surface. A PEG-modified liposome that retains is one embodiment. Such PEG-modified liposomes exhibiting retention in blood have the characteristic of being easily leaked and accumulated from the blood into the target tissue, for example, cancer tissue, due to the EPR (enhanced permeability and retention) effect. Has been reported ( ).

PEG-modified liposomes encapsulating doxorubicin utilizing the above properties have already been marketed in Europe and the United States for indications of Kaposi's sarcoma and breast cancer, and their usefulness has been reported in terms of reducing side effects and therapeutic effects
). However, in this technique, the retention in the circulating blood is maintained, but the presence of the water-soluble polymer serves as a barrier, and as a result, the action of the drug on the organ, the approach to the cell, the contact, etc. are suppressed, Phenomena in which the delivery of drugs into cells is impaired, such as a phenomenon in which the drug concentration at the action point does not increase and a phenomenon in which the drug efficacy does not increase despite the large amount of liposomes distributed, have been recognized. For example, from the results of studies using tumor-bearing mice, the accumulation property of drug-encapsulated PEG-modified liposomes does not necessarily correlate with the antitumor effect, that is, the antitumor effect is not always high even if the accumulation property in tumor is high Has been reported ( ).

Under such circumstances, several reports have been made on means for enhancing the drug efficacy of a drug encapsulated in liposomes in a target organ and cells while maintaining retention in circulating blood. As one of them, a method has been reported in which a hydrophilic polymer or the like acts effectively at the time of administration, and the water-soluble polymer is desorbed in the vicinity of the target cell. For example,
Discloses a liposome having on its surface an affinity moiety effective to specifically bind to a biological surface and a hydrophilic polymer chain effective to shield the affinity moiety.

According to this method, until the retention in the circulating blood is achieved, the hydrophilic polymer chain on the liposome surface functions effectively, and thereafter, by administering a release agent for releasing the hydrophilic polymer chain, The hydrophilic polymer chain is released and the shielded affinity moiety functions, thereby exhibiting specific binding properties to the cell surface. However, in this method, it is necessary to administer a free agent such as cysteine or ascorbate in order to release the hydrophilic polymer (chain). In actual use, there is a concern about the effect of the free agent in vivo. There remains a problem with the safety of the release agent.

Also,
Discloses a technique capable of changing the properties of liposomes by detaching water-soluble polymers from the surface of the liposomes over time after administration to a living body without depending on environmental differences. further, Discloses a PEG-modified fusogenic liposome that can regulate the rate of membrane fusion with the cell membrane of the target cell by changing the alkyl chain length of the lipid part of the conjugate of polyethylene glycol and lipid (lipid that forms the liposome) Has been. However, in these technologies, the water-soluble polymer is detached before the liposome reaches the disease site, and the retention in the circulating blood may not be maintained, and there is still room for improvement. .

on the other hand,
, Discloses a liposome as a drug carrier using polyethylene glycol cholesteryl ether for the purpose of avoiding liposome entrapment (RES system) by phagocytes in the liver and spleen. After intravenous administration to rats, the liposomes showed retention in the circulating blood avoiding the RES system. However, it is described in any of the above-mentioned documents that polyethylene glycol cholesteryl ether is a liposome-forming base having an attribute that achieves improved delivery to cells or polyethylene glycol cholesteryl ether achieves improved delivery to cells. There is no suggestion.

JP-T-2001-503396 Japanese Patent Laid-Open No. 10-287554 T M Allen, A Chung, “Large Unilamella Liposomes with Low Uptake into the Reticulo Endocerial System”, Feves Letter, 1987, vol. 223, p.42-46 (TM Allen and A. Chonn, “Large unilamellar liposomes with low uptake into the reticuloendothelial system”, FEBS Letter, 1987, vol. 223, p.42-46) Alexander El Krivanov, Kazuo Murayama, Hurademir Pee Tolhirin, Reef Fan, “Amphipathic Polyethylene Glycols Effectively Pro Long The Circulation Time of Liposomes”, Feves Letter, 1990, vol. 268, p.235-237 (Alexander) L. Klibanov, Kazuo Maruyama, Vladimir P. Torchilin and Leaf Huang, “Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes”, FEBS Letter, 1990, vol. 268, p.235-237) Takefumi Takeuchi, Hiroyuki Kojima, Hiromitsu Yamamoto, Yoshiaki Kawashima, “Surface Modification (Coating) of Liposomes with Various Partially Hydrophobized Water-soluble Polymers and Evaluation of Their Retention in Blood”, Pharmacology, 2001, vol.61, p. 86-96 Yokoyama, Okano, “Targeting of Anti-Cancer Drug With Nanosized Carrier System”, Japanese Clinical, Japanese Journal of Clinical Medicine, 1998, vol. 56, p.3227-34 (Yokoyama M. Okano T. “Targeting of anti-cancer drugs with nano-sized carrier system ”. Nippon Rinsho-Japanese Journal of Clinical Medicine. 1998, vol. 56, p.3227-34) Cokel, Spencer, "Polyethylene glycol-liposomal doxorubicin. Review of It's pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy in the management of AIDS related Kaposi Sarcoma" Drugs, 1997, vol. 53, p.520-38, (Coukell AJ. Spencer CM. “Polyethylene glycol-liposomal doxorubicin. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy in the management of AIDS-related Kaposi's sarcoma.” Drugs. 1997, vol. 53 , p.520-38) Hong, Han, Tseng, Pan, Chen, Liu, Chang, “Direct Comparison of Liposomal Doxorubicin with or Without Polyethylene Glycol Coating in C-26 Tumor Bearing Mys: Is Surface Coating with Polyethylene Glycol Beneficial?” Clinical Cancer Research. 1999, vol. 5, p.3645-52. (Hong RL. Huang CJ. Tseng YL. Pang VF. Chen ST. Liu JJ. Chang FH. ≡Direct comparison of liposomal doxorubicin with or without polyethylene glycol coating in C-26 tumor-bearing mice: is surface coating with polyethylene glycol beneficial ? ≡. Clinical Cancer Research. 1999, vol. 5, p.3645-52) Jintanjali Adrakha-Hatcheon, Marcel Bee. Barry, Clifford Earl. Shoe and Thomas D. Madden, “Controlled Destabilization of a Liposomal Drug Delivery System Enhancements Mitoxantrone Anti-Tummer Activity”, Nature Biotechnology, 1999, vol. 17, p.775-779. (Gitanjali Adlakha-Hutcheon, Marcel B. Bally, Clifford R. Shew & Thomas D. Madden, “Controlled destabilization of a liposomal drug delivery system enhances mitoxantrone antitumor activity”, Nature Biotechnology, 1999, vol. 17, p.775-779 ) Hideaki Ishiwata, Arin Feltut-Doi, Hiroo Takeo, and Koichirou Miyajima, “Physical-Chemistry Characteristics and Biodistribution of Poly (ethylene glycol) -Coated Liposomes Using Poly (oxyethylene) cholesteryl ether”, Chem Farm Bull, 1995, 43 (6), p.1005-1011. (Hideki ISHIWATA, Aline Vertut-Doi, Takeo HIROSE, and Koichiro MIYAJIMA, “Physical-Chemistry Characteristics and Biodistribution of Poly (ethylene glycol)-Coated Liposomes Using Poly (oxyethylene) Cholesteryl Ether”, Chem. Pharm. Bull. 1995, 43 (6), p.1005-1011. Hideaki Ishiwata, Satoshi B. Sato, Sato Kobayashi, Masahide Ok, Allin Feltut-Doi, and Koichiro Miyajima, “Poly (Ethylene Glycol) Derivatives of Cholesterol Reduces Binding Step of Liposome Uptake by Muline Macrophage-Like Cell Line Jpa Toma Cell Line Hepsey Two ", Chem Farm Bull, 1998, 46 (12), p.1907-1913. (Hideki ISHIWATA, Satoshi B. SATO, Satoe KOBAYASHI, Masahide OKU, Aline Vertut-Doi, and Koichiro MIYAJIMA, “Poly (ethylene glycol) Derivative of Cholesterol Reduces Binding Step of Liposome Uptake by Murine Macrophage-like Cell Line J774 and Human Hepatoma (Cell Line HepG2 ”, Chem. Pharm. Bull., 1998, 46 (12), p. 1907-1913.)

An object of the present invention is to provide a liposome having excellent retention in the circulating blood and delivery within the target cell, and a drug comprising the liposome, which does not require any special operation such as administration of a release agent. It is to provide a target intracellular delivery improver.

Under such circumstances, the present inventors have conducted various studies on the target organ of the drug encapsulated in the liposome and a method for enhancing the drug delivery property into the cell. It has been found that polyethylene glycol cholesteryl ether, which has only been clarified, has the effect of enhancing the drug efficacy in target organs and cells. Liposomes with polyethylene glycol diacylglycerol ester as one of the lipid membrane constituents in addition to polyethylene glycol cholesteryl ether also show retentivity in circulating blood, and surprisingly, an aqueous drug solution can be used as a target cell. As a result, the present invention was completed.

According to the present invention, retention of liposomes in the circulating blood can be maintained, and the drug efficacy of the drug in target organs and cells can be enhanced.
That is, the present invention
1. An agent for improving intracellular delivery of a drug comprising a liposome prepared using polyethylene glycol cholesteryl ether as part of a liposome-forming base;
2. The agent for improving the intracellular delivery of a drug according to the above 1, wherein the polyethylene glycol cholesteryl ether has an average molecular weight of polyethylene glycol of 200 to 20000,

3. The agent for improving the intracellular delivery of a drug according to the above 1, further comprising a polyethylene glycol diacylglycerol ester as part of a liposome-forming base,
4). The polyethylene glycol diacylglycerol ester is one selected from the group consisting of polyethylene glycol distearyl glycerol ester, polyethylene glycol dipalmitoyl glycerol ester, polyethylene glycol dimyristoyl glycerol ester, polyethylene glycol dilauryl glycerol ester, and polyethylene glycol dioleyl glycerol ester Or an agent for improving target intracellular delivery of the drug according to 3 above, which is at least two kinds
5. The agent for improving the intracellular delivery of a drug according to the above 3, wherein the polyethylene glycol diacylglycerol ester has an average molecular weight of polyethylene glycol of 200 to 20000,

6). 4. The target intracellular delivery improver according to 3 above, wherein the molar ratio of polyethylene glycol cholesteryl ether to polyethylene glycol diacylglycerol ester is 1: 100 to 100: 1.
7). A drug-containing liposome containing a drug, polyethylene glycol cholesteryl ether, and polyethylene glycol diacylglycerol ester,
8). The drug-containing liposome according to 7 above, wherein the average particle size is 400 nm or less,
About.

The present invention also relates to the use of polyethylene glycol cholesteryl ether for producing an agent for improving the intracellular delivery of a target drug. Use of the above polyethylene glycol cholesteryl ether, wherein polyethylene glycol has an average molecular weight of 200 to 20000. About. The polyethylene glycol diacylglycerol ester is an embodiment further comprising a polyethylene glycol diacylglycerol ester, a polyethylene glycol distearyl glycerol ester, a polyethylene glycol dipalmitoyl glycerol ester, a polyethylene glycol dimyristoyl glycerol ester, a polyethylene glycol dilauryl glycerol ester, and Use of one or more selected from the group consisting of polyethylene glycol cholesteryl ether, which is a polyethylene glycol dioleyl glycerol ester, polyethylene glycol cholesteryl ester having an average molecular weight of polyethylene glycol of polyethylene glycol diacyl glycerol ester of 200 to 20000 The use of ether, and further the molar ratio of polyethylene glycol cholesteryl ether and polyethylene glycol diacyl glycerol esters is 1: 100-100: relates to the use of polyethylene glycol cholesteryl ether is 1.

On the other hand, the present invention relates to a method for improving target intracellular delivery of a drug by liposome, characterized in that polyethylene glycol cholesteryl ether is part of the liposome-forming base. The above improvement method having an average molecular weight of 200 to 20000, the method for improving target intracellular delivery of a drug further containing polyethylene glycol diacylglycerol ester, polyethylene glycol diacylglycerol ester is polyethylene glycol distearyl glycerol ester, polyethylene glycol dipalmitoyl glycerol ester , Polyethylene glycol dimyristoyl glycerol ester, polyethylene glycol dilauryl glycerol ester Ether, and to a polyethylene glycol dioleyl glycerol esters of one or targeted intracellular delivery of improved methods of drug is 2 or more elements are selected from the group consisting of. Furthermore, the present invention relates to the above improved process wherein the polyethylene glycol diacylglycerol ester has an average molecular weight of polyethylene glycol of 200 to 20000, and the molar ratio of polyethylene glycol cholesteryl ether to polyethylene glycol diacylglycerol ester is 1: 100-100. The present invention relates to a method for improving target intracellular delivery of a drug having a ratio of 1.

As yet another aspect, the present invention relates to a drug-containing liposome with improved target intracellular delivery of a drug comprising a drug, polyethylene glycol cholesteryl ether, and polyethylene glycol diacylglycerol ester, and has an average particle size. The present invention relates to the above-mentioned drug-containing liposome having a wavelength of 400 nm or less, and further to the above-mentioned drug-containing liposome, wherein the drug is a cationic drug, and the present invention relates to the above-mentioned drug-containing liposome, wherein the drug is an anticancer agent or an antibacterial agent It is.

The drug in the present invention means a drug that interacts with in vivo molecules present in cells and exhibits a medicinal effect. The specific drug is not particularly limited, but a cationic compound is preferable. This is because it is advantageous for encapsulation in liposomes. For example, doxorubicin, cisplatin, irinotecan, fluorouracil, carmofur, aclarubicin hydrochloride, daunorubicin hydrochloride, doxorubicin hydrochloride, mitomycin, paclitaxel, epirubicin hydrochloride, idarubicin hydrochloride, pirarubicin hydrochloride, bleomycin, pepromycin sulfate, etoposide hydrochloride, irinotecan hydrochloride, irinotecan hydrochloride, Examples thereof include nucleic acid drugs such as docetaxel, vincristine sulfate, vindesine sulfate, binplastin sulfate, tamoxifen citrate, schizophyllan, krestin, gefitinib, cisplatin, cyclophosphamide, thiotepa, gene, antisense, and siRNA. Moreover, it is not limited to one drug, and it is possible to use a combination of several kinds of drugs as desired.

  In the present invention, improvement in intracellular delivery means increasing the amount of a drug transferred into a cell or increasing the amount of a compound that reaches an action point after the compound enters the cell, Various measurement methods make it possible to know the improvement of delivery. For example, in the case of doxorubicin or the like, it can be confirmed by measuring the cell killing effect by the cytotoxicity test shown below. Such an effect can be achieved by liposomes prepared with polyethylene glycol cholesteryl ether, preferably polyethylene glycol diacylglycerol ester as part of the liposome-forming base.

The term “polyethylene glycol cholesteryl ether” means a polyethylene glycol (PEG) or a PEG derivative having a hydroxyl group bonded to a cholesterol 3-position hydroxyl group by an ether bond. The average molecular weight (weight average molecular weight) of the PEG moiety of polyethylene glycol cholesteryl ether is preferably in the range of 200-20000, more preferably in the range of 400-5000, and most preferably in the range of 1000-2000. It is. This is because as the molecular weight of the PEG moiety becomes larger than that of cholesteryl ether, which is an anchor to the liposome, it becomes easier to detach from the liposome, and as a result, the effect cannot be exhibited.

By using polyethylene glycol diacylglycerol ester in addition to polyethylene glycol cholesteryl ether at the time of liposome formation, the drug efficacy can be further enhanced. The polyethylene glycol diacylglycerol ester is selected from the group consisting of polyethylene glycol distearyl glycerol ester, polyethylene glycol dipalmitoyl glycerol ester, polyethylene glycol dimyristoyl glycerol ester, polyethylene glycol dilauryl glycerol ester, and polyethylene glycol dioleyl glycerol ester. Species or two or more can be used. An appropriate one is selected according to the other phospholipids constituting the lipid membrane of the liposome, the kind of cholesterol, the kind of polyethylene glycol cholesteryl ether, and the blending ratio thereof. The average molecular weight (weight average molecular weight) of the PEG portion of the polyethylene glycol diacylglycerol ester is preferably in the range of 200-20000, more preferably in the range of 400-5000, and most preferably in the range of 1000-2000. Is preferred.

The molar ratio of polyethylene glycol cholesteryl ether to polyethylene glycol diacylglycerol ester in the present invention is 1: 100-100: 1, preferably 1: 5-5: 1, most preferably 1: 2-2: 1. This is because if the amount of polyethylene glycol diacylglycerol ester is decreased, the blood stability of the liposome itself deteriorates, and as a result, the ability to reach the target cells is lost, and the intracellular delivery effect cannot be exerted in vivo. . Moreover, it is because a cell delivery effect will attenuate when the compounding quantity of polyethyleneglycol cholesteryl ether decreases.

When preparing the liposome of the present invention, phospholipid, cholesterol and the like generally used for preparing liposomes are blended. In the present invention, “as part of the liposome-forming base” means a phospholipid as shown below other than polyethylene glycol cholesteryl ether and polyethylene glycol diacylglycerol ester within the range not affecting the effects of the present invention. It means that cholesterol and the like can be blended. The “liposome-forming base” in the present invention means a base necessary for preparing liposomes. Phospholipids include egg yolk lecithin, soybean lecithin, hydrogenated lecithin, distearyl phosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, dilaurylphosphatidylcholine, dioleoylphosphatidylcholine, Distearyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dilauryl phosphatidylglycerol, distearyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dimyristoyl phosphatidylethanol Amines, dilauryl phosphatidylethanolamine, distearyl phosphatidic acid, dipalmitoyl phosphatidic acid, Myristoyl phosphatidic acid, such as dilauryl phosphatidic acid. One or more of these can be selected.

The particle size of the liposome of the present invention is not particularly limited as long as it can achieve the effect of the present invention, but when administered intravenously, the average particle size is desirably 400 nm or less. This is because liposomes with a particle size exceeding 400 nm are taken up by the reticuloendothelial system such as the liver and spleen and the lungs and have poor retention in the blood, so that they are not easily transferred to the organ where the action point exists. .

The liposome of the present invention is prepared by applying a production method widely used in the technical field of the liposome preparation. In preparing the liposome, specifically, for example, the Bangham method, the freeze-thaw method, the ethanol injection method, the reverse phase evaporation method, and the Methods in Enzymology 367 described in “Nojima, Liposomes, Nanedo (1988)” are used. 99-110 (2003), or a lyophilization method, a pH gradient method, or the like can be applied.

For example, as described in Examples 1 and 2, polyethylene glycol cholesteryl ether, if necessary, further polyethylene glycol diacylglycerol ester, and any liposome-forming base such as phospholipids such as distearyl phosphatidylcholine Is dissolved or suspended in a solvent at a predetermined ratio, and the solvent is distilled off. A buffering agent and a stabilizer are added to this to hydrate, and after substitution with nitrogen, sonication or the like is applied to prepare a liposome dispersion. When adjusting the liposome particle size, the Extrusion method or the like is further applied.

By employing polyethylene glycol cholesteryl ether, preferably polyethylene glycol diacylglycerol ester, the liposome of the present invention having improved delivery to target cells is provided. The liposome is administered to a living body by injection, oral administration, pulmonary administration, nasal administration, or other administration methods. Such liposomes may contain excipients and the like as long as the effects of the present invention are not impaired. Examples of excipients include pharmaceutically acceptable salts, surfactants, saccharides, amino acids, organic acids, and other water-soluble substances.

Specific salts include potassium L-glutamate, sodium L-glutamate, sodium edetate, sodium caprylate, sodium carbazochrome sulfonate, sodium carboxymethylcellulose, sodium citrate, calcium gluconate, sodium gluconate, magnesium gluconate, Sodium metasulfobenzoate, sodium monohydrogen phosphate, sodium dihydrogen phosphate, dipotassium phosphate, potassium dihydrogen phosphate, aluminum chloride, potassium chloride, calcium chloride, sodium chloride, sodium acetate, sodium carbonate, sodium bicarbonate, etc. Examples of the surfactant include sorbitan sesquioleate, sorbitan fatty acid ester, polyoxyethylene (160) polyoxypropylene (30) glycol Polyoxyethylene sorbitan monolaurate, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, polyoxyethylene hydrogenated castor oil 50, polyoxyethylene hydrogenated castor oil 60, polysorbate 20, polysorbate 80, macrogol 400, macrogol 4000 Macrogol 600 and the like, and saccharides include D-sorbitol, D-mannitol, inositol, xylitol, dextran, glucose, maltose, lactose, sucrose and the like, and amino acids include methionine, aspartic acid, alanine , Arginine, glycine, cysteine, taurine, histidine, phenylalanine, glutamic acid, lysine, etc., and organic acids include citric acid, succinic acid, malic acid, lactic acid, etc. It is, as the other water-soluble materials, ascorbic acid, human serum albumin, sodium chondroitin sulfate, gelatin, hydrolyzed gelatin, heparin sodium, and the like.

The result of the cell killing effect with respect to P388 leukemia cells of the doxorubicin aqueous solution and the liposomal formulation of doxorubicin is shown. The result of the drug uptake | capture to the Ehrlich ascites tumor cell of the liposomal formulation of doxorubicin aqueous solution and doxorubicin is shown. The result of the antitumor effect (tumor weight) in the P388 leukemia cell transplant mouse | mouth of the doxorubicin aqueous solution and the liposomal formulation of doxorubicin is shown. The result of the tumor doxorubicin density | concentration in the P388 leukemia cell transplant mouse | mouth of the liposomal formulation of doxorubicin is shown.

EXAMPLES The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited thereby.

Example 1
Distearyl phosphatidylcholine (DSPC), cholesterol (Chol), distearyl phosphatidylglycerol (DSPG), polyethylene glycol cholesteryl ether (PEG-Chol) having an average molecular weight of 2000, respectively, 100 μmol: 100 μmol: 60 μmol: Dissolve 15 μmol and doxorubicin 18 μmol in chloroform / methanol mixture (4: 1, v / v), distill off the organic solvent under reduced pressure with a rotary evaporator under a nitrogen gas stream, create a lipid film on the inner wall of the eggplant flask, and further desiccator The solvent was completely distilled off using a vacuum pump. To this, 10 ml of 9.0% sucrose / 10 mM lactate buffer (pH 4.0) was added, hydrated at 75 ° C., purged with nitrogen, and sonicated to obtain a liposome dispersion. Furthermore, the particle size of the liposome was adjusted by the Extrusion method (PEG-CHO (2000) -LDOX).

Example 2
DSPC, Chol, DSPG, polyethylene glycol cholesteryl ether (PEG-Chol) having an average molecular weight of 2000 of polyethylene glycol, and polyethylene glycol distearyl glycerol ester (PEG-DSG) having an average molecular weight of 2000 of polyethylene glycol are 100 μmol, 100 μmol, 60 μmol, respectively. 7.5 μmol, 7.5 μmol, and 18 μmol of doxorubicin were dissolved in a chloroform / methanol mixture (4: 1, v / v), and the same procedure as in Example 1 was performed to prepare a liposome dispersion liquid having an adjusted particle size.
(PEG- (DSG (2000): CHO (2000) = 1: 1) -LDOX).

Example 3
DSPC, Chol, DSPG, polyethylene glycol cholesteryl ether (PEG-Chol) having an average molecular weight of 2000 of polyethylene glycol, and polyethylene glycol distearyl glycerol ester (PEG-DSG) having an average molecular weight of 2000 of polyethylene glycol are 100 μmol, 100 μmol, 60 μmol, respectively. 3.75 μmol, 11.25 μmol, and 18 μmol of doxorubicin were dissolved in a chloroform / methanol mixture (4: 1, v / v), and the same procedure as in Example 1 was performed to prepare a liposome dispersion liquid having an adjusted particle size.
(PEG- (DSG (2000): CHO (2000) = 3: 1) -LDOX).

Comparative Example 1
Liposomes adjusted for particle size by dissolving DSPC, Chol and DSPG in 100 μmol, 100 μmol, 60 μmol and 18 μmol doxorubicin in chloroform / methanol mixture (4: 1, v / v), respectively, and operating in the same manner as in Example 1. A dispersion was prepared (PLDOX).

Comparative Example 2
DSPC, Chol, DSPG, polyethylene glycol distearylglycerol ester (PEG-DSG) having an average molecular weight of 2000, 100 μmol, 100 μmol, 60 μmol, 15 μmol, and 18 μmol of doxorubicin, respectively, in a chloroform / methanol mixture (4: 1, v / v ) And the same operation as in Example 1 was performed to prepare a liposome dispersion liquid (PEG-DSG (2000) -LDOX) having an adjusted particle size.

Comparative Example 3
18 μmol of doxorubicin was dissolved in 10 ml of 9.0% sucrose / 10 mM lactic acid buffer (pH 4.0) to obtain an aqueous solution of doxorubicin (DOX sol).

Experimental example 1
Ehrlich ascites tumor cells (1 × 10 ⁶ ) were added with solutions prepared in Examples 1, 2 and Comparative Examples 1, 2 and 3 adjusted to various concentrations, and the cell killing effect was examined using WST. .
From the results of plotting the relationship between the doxorubicin concentration in the preparation and the cell killing effect, the doxorubicin concentration at which 50% cell killing effect was obtained was calculated (Table 1). From this, it was shown that the liposome preparation of the present invention shows a remarkable pharmacological effect as compared with the comparative example.

Table 1. Cytocidal effect of liposomal formulations of doxorubicin and doxorubicin on Ehrlich ascites tumor cells

Experimental example 2
P388 leukemia cells (1 × 10 ⁶ ) were prepared by adding the liquid prepared by preparing the preparations prepared in Examples 1 and 2 and Comparative Examples 1, 2 and 3 so that the concentration of doxorubicin was 0.01 μg / ml. The cell killing effect was examined using WST in the same manner as described above. The ratio of the number of viable cells is shown in FIG. Thus, the liposome preparation of the present invention showed a remarkable pharmacological effect as compared with Comparative Examples 1 and 2, and showed the same effect as the aqueous solution of doxorubicin of Comparative Example 3 as a positive control.

Experimental Example 3
To P388 leukemia cells (1 × 10 ⁶ ), solutions prepared in Examples 1, 2, 3 and Comparative Example 2 adjusted to various concentrations were added, and the cell killing effect was examined using WST.
From the results of plotting the relationship between the doxorubicin concentration in the preparation and the cell killing effect, the doxorubicin concentration at which 50% cell killing effect was obtained was calculated (Table 2). From this, it was shown that the liposome preparation of the present invention shows a remarkable pharmacological effect as compared with the comparative example.
Table 2 Cytotoxic effects of liposomal preparations of doxorubicin and doxorubicin on P388 leukemia cells

Experimental Example 4
To the Ehrlich ascites tumor cells, the liquid prepared by adjusting the concentration of doxorubicin to 5.0 μg / ml was added to the preparations prepared in Examples 1 and 2 and Comparative Examples 1 and 2, and the drug uptake into the cells over time was examined.
As a result of examining the time transition of doxorubicin concentration per a certain number of cells, it was shown that when the liposome preparation of the present invention was used, significant drug uptake was obtained as compared with the comparative example (FIG. 2).

Experimental Example 5
P388 leukemia cells (5.0 × 10 ⁵ ) were planted subcutaneously on the back of the mice to form solid tumors. On the 5th, 8th, and 11th days after planting, tail vein injection of the preparations prepared in the control (saline solution), Comparative Example 1 and Example 3 to 2.5 mg / kg as a single dose of doxorubicin. (Total dose 7.5 mg / kg). On day 13 after planting (48 hours after the third administration), the tumor was excised, and the tumor weight and the concentration of doxorubicin in the tumor cells were measured. Tumor weight was significantly reduced in Example 3 (FIG. 3), and the concentration of doxorubicin in the tumor was also increased (FIG. 4).

The present invention provides a drug target that significantly improves the intracellular delivery of the drug by liposomes in the target cell while maintaining the retentivity in the circulating blood without requiring a special operation such as administration of a release agent. It is useful as an agent for improving intracellular delivery and the liposome.

Claims (8)

An agent for improving target intracellular delivery of a drug comprising a liposome prepared using polyethylene glycol cholesteryl ether as a part of a liposome-forming base. The target intracellular delivery improver for a drug according to claim 1, wherein the polyethylene glycol cholesteryl ether has an average molecular weight of polyethylene glycol of 200 to 20,000. The agent for delivering a drug into a target cell according to claim 1, further comprising polyethylene glycol diacylglycerol ester as a part of the liposome-forming base. The polyethylene glycol diacylglycerol ester is one selected from the group consisting of polyethylene glycol distearyl glycerol ester, polyethylene glycol dipalmitoyl glycerol ester, polyethylene glycol dimyristoyl glycerol ester, polyethylene glycol dilauryl glycerol ester, and polyethylene glycol dioleyl glycerol ester Or the target intracellular delivery improvement agent of the drug of Claim 3 which is 2 or more types. The agent for improving intracellular delivery of a drug according to claim 3, wherein the polyethylene glycol diacylglycerol ester has an average molecular weight of polyethylene glycol of 200 to 20,000. The target intracellular delivery improver according to claim 3, wherein the molar ratio of polyethylene glycol cholesteryl ether to polyethylene glycol diacylglycerol ester is 1: 100-100: 1. A drug-containing liposome containing a drug, polyethylene glycol cholesteryl ether, and polyethylene glycol diacylglycerol ester. The drug-containing liposome according to claim 7, having an average particle size of 400 nm or less.

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